Method for producing low-molecular-weight compound

Abstract

Provided is a method for producing a low-molecular-weight compound, the method involving isolating the low-molecular-weight compound contained in a culture supernatant of algae.

Description

Method for producing low molecular weight compounds

[0001] This disclosure relates to a method for producing low molecular weight compounds.

[0002] Microalgae are attracting attention in aquaculture as a sustainable biological resource, and their use as feed for farmed fish and shellfish is progressing (Non-Patent Literature 1). Furthermore, because microalgae contain a variety of substances, there is hope that they can be used to produce substances in an environmentally friendly way (Non-Patent Literature 2).

[0003] Conventional algal utilization techniques include extracting lipids from the harvested cells and using the remaining components as protein for aquaculture feed (Non-Patent Document 2), and harvesting the entire algal cell and using it mainly as a substitute for fish oil (Non-Patent Document 3).

[0004] However, these current technologies still have challenges. For example, algae secrete useful substances such as polysaccharides outside their cells (Non-Patent Literature 4), but current technologies only collect the cells, and the useful substances contained in the supernatant are not fully utilized. Furthermore, a technology to control the amount of useful substances produced in the supernatant has not yet been established. By solving these problems, it is expected that an environmentally friendly method of producing substances using algae will be established, the utilization efficiency of algae will be increased, and their value as aquaculture feed will be further enhanced.

[0005] Fukada, Y. et al. (2021). Effects of complete replacement of fish oil with plant oil mixtures and algal meal on growth performance and fatty acid composition in juvenile yellowtail Seriola quinqueradiata. Nippon Suisan Gakkaishi, 87(4), 314. Sarker, PK. et al., Towards sustainable aquafeeds: Evaluating substitution of fishmeal with lipid-extracted microalgal co-product (Nannochloropsis oculata) in diets of juvenile Nile tilapia (Oreochromis niloticus), PLoS One. 2018 Jul 31;13(7):e0201315. Sarker, PK. Towards sustainable aquafeeds: Complete substitution of fish oil with marine microalga Schizochytrium sp. Improves growth and fatty acid deposition in juvenile nile tilapia (Oreochromis niloticus), PLoS One. 2016; 11(6): e0156684. Smestad, B. et al., Production of carbohydrates by the marine diatom Chaetoceros affinis var. willei (Gran) Hustedt. II. Preliminary investigation of the extracellular polysaccharide, Journal of Experimental Marine Biology and Ecology, Volume 9, Issue 2, 1972, Pages 125-136.

[0006] This disclosure is made to solve the problems described above and aims to provide a method for producing low molecular weight compounds.

[0007] One aspect of the present disclosure is a method for producing a low molecular weight compound, comprising isolating the low molecular weight compound contained in the culture supernatant of algae, wherein the low molecular weight compound is any of the following: kynurenine, topiramate, inosine, ornithine, ATP, carnitine, choline, cystine, Met, Thr, pyridoxal, pyridoxine, Trp, norspermidine, spermidine, Val, Leu, Phe, creatine, glycolic acid, capryloylglycine, 1-methyladenine, 1-methyladenosine, 2'-deoxyadenosine, 5'-deoxyadenosine, 2'-deoxycytidine, 2'-deoxyguanosine, 2-hydroxyoctanoic acid, 2-oxoglutaric acid, 3-methylcytidine, 2'-O-methylcytidine, 3-sulfopropanediol, 4-guanidinobutyric acid acid, 4-hydroxyquinoline, 4-oxopyrrolidine-2-carboxylic acid, 5'-deoxy-5'-methylthioadenosine, 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylic acid, 5-methylcytidine, 7-methylguanine, adenine, adenosine, Ala, Ala-Ala, anthranilic acid, argininosuccinic acid, Asn, Asp, cis-aconitic acid, creatinine, cystathionine, cytidine, ethanolamine, galacturonic acid, glucuronic acid, Gln, Glu, gluconolactone, glutathione, GSSG, Gly, Gly-Asp, Gly-Leu, guanine, guanosine, H-Asp(Gly-OH)-OH,homoserine, Ile, m-hydroxybenzoic acid, p-hydroxybenzoic acid, melamine, methionine sulfoxide, mevalolactone, morpholine, mucic acid, N-acetylgalactosamine, N-acetylglucosamine, N-acetylmannosamine, N-acetyllysine, N-acetylornithine, N-acetylputrescine, N, 1 ,N 12 -diethylspermine, N 1 -methylguanosine, N 2 -methylguanine, N 6 -methyladenine, N 6 -methyladenosine, N 8 -acetylspermidine, nicotinic acid, O-acetylcarnitine, p-aminobenzoic acid, piperidine, Pro, pyrophosphate, Ser, Ser-Glu, threonic acid, thymidine, thymine, Tyr-Arg, Tyr-Glu, uracil, uridine, urocanic acid, β-hydroxyisovaleric acid, 2-hydroxyvaleric acid, 2-hydroxyisovaleric acid.

[0008] According to this disclosure, a method for producing low molecular weight compounds can be provided.

[0009] Changes in cell density in Chaetoceros gracilis cultures from the start of culture to day 10. Black circles indicate cell density at 25°C, and white circles indicate cell density at 30°C.

[0010] The following describes non-limiting embodiments of this disclosure. This disclosure is not limited to the embodiments described below.

[0011] The inventors have newly discovered that useful substances such as kynurenine and topiramate accumulate in the culture supernatant of algae, which are known as raw materials for aquaculture feed, as the algae grow. Based on this discovery, this disclosure provides a method for producing low molecular weight compounds using algae. This method makes it possible to effectively utilize the culture supernatant, which was conventionally discarded as residue, for the production of useful substances.

[0012] The molecular weight of the "low molecular weight compound" in this disclosure may be 1000 g / mol or less, 900 g / mol or less, or 800 g / mol or less. The "low molecular weight compound" may also be an organic compound. The "low molecular weight compound" in this disclosure may be a compound with a molecular weight of 800 g / mol or less, or an organic compound with a molecular weight of 800 g / mol or less.

[0013] This disclosure provides a method for producing low molecular weight compounds. The method for producing low molecular weight compounds includes isolating low molecular weight compounds contained in the culture supernatant of algae. In the method for producing the low molecular weight compound of the embodiment, the low molecular weight compound is one of the following: kynurenine, topiramate, inosine, ornithine, ATP, carnitine, choline, cystine, Met, Thr, pyridoxal, pyridoxine, Trp, norspermidine, spermidine, Val, Leu, Phe, creatine, glycolic acid, capryloylglycine, 1-methyladenine, 1-methyladenosine, 2'-deoxyadenosine, 5'-deoxyadenosine, 2'-deoxycytidine, 2'-deoxyguanosine, 2-hydroxyoctanoic acid, 2-oxoglutaric acid, 3-methylcytidine, 2'-O-methylcytidine, 3-sulfopropanediol, 4-guanidinobutyric acid, 4-hydroxyquinoline, 4-oxopyrrolidine-2-carboxylic acid, 5'-deoxy-5'-methylthioadenosine, 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylic acid, 5-methylcytidine, 7-methylguanine, adenine, adenosine, Ala, Ala-Ala, anthranilic acid, argininosuccinic acid, Asn, Asp, cis-Aconitic acid, creatinine, cystathionine, cytidine, ethanolamine, galacturonic acid, glucuronic acid, Gln, Glu, gluconolactone, glutathione, GSSG, Gly, Gly-Asp, Gly-Leu, guanine,guanosine, H-Asp(Gly-OH)-OH, homoserine, Ile, m-hydroxybenzoic acid, p-hydroxybenzoic acid, melamine, methionine sulfoxide, mevalolactone, morpholine, mucic acid, N-acetylgalactosamine, N-acetylglucosamine, N-acetylmannosamine, N-acetyllysine, N-acetylornithine, N-acetylputrescine, N, 1 ,N 12 -diethylspermine, N 1 -methylguanosine, N 2 -methylguanine, N 6 -methyladenine, N 6 -methyladenosine, N 8-acetylspermidine, nicotinic acid, O-acetylcarnitine, p-aminobenzoic acid, piperidine, Pro, pyrophosphate, Ser, Ser-Glu, threonic acid, thymidine, thymine, Tyr-Arg, Tyr-Glu, uracil, uridine, urocanic acid, β-hydroxyisovaleric acid, 2-hydroxyvaleric acid, 2-hydroxyisovaleric acid. For those of these low molecular weight compounds that are listed by abbreviation, the corresponding names are shown in parentheses in the following list: ATP (adenosine triphosphate), Met (methionine), Thr (threonine), Trp (tryptophan), Val (valine), Leu (leucine), Phe (phenylalanine), Asn (asparagine), Asp (aspartic acid), Gln (glutamine), Glu (glutamic acid), Gly (glycine), Ile (isoleucine), Pro (proline), Ser (serine), Ala-Ala (alanylalanine), Gly-Asp (glycylaspartic acid), Gly-Leu (glycylleucine), H-Asp(Gly-OH)-OH (asparagylglycine; 2-amino-4-(carboxymethylamino)-4-oxo-butanoic acid), GSSG (oxidized glutathione), Ser-Glu (cerylglutamic acid), Tyr-Arg (tyrosylarginine), Tyr-Glu (tyrosylglutamic acid).

[0014] The low molecular weight compound in the embodiment is one or more of the following: kynurenine, topiramate, inosine, ornithine, ATP, carnitine, choline, cystine, Met, Thr, pyridoxal, pyridoxine, Trp, norspermidine, spermidine, Val, Leu, Phe, creatine, glycolic acid, or capryloylglycine. In another embodiment, the low molecular weight compound is kynurenine and / or topiramate. In yet another embodiment, the low molecular weight compound is kynurenine. Kynurenine is known as a stress-reducing substance in fish (Ikari, T. et. al., (2023). Kynurenine promotes Calcitonin secretion and reduces cortisol in the Japanese flounder Paralichthys olivaceus. Scientific Reports, 13(1), Japanese Patent No. 7093961). In yet another embodiment, the low molecular weight compound is topiramate. Topiramate is known as a raw material for antiepileptic drugs (Fariba KA, Saadabadi A. Topiramate. 2024 Jun 8. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan. PMID: 32119417.).

[0015] The types of algae in this disclosure are not limited. The algae may be non-sessile algae. The algae may be, for example, unicellular algae or microalgae. Examples of algae that may be used include any of the following classifications: Aurantiochytrium, Chlamydomonas, Chlorella, red algae Schizoan, Spirulina, Botryococcus, Euglena, haptophytes, Prasinophytes, green algae, brown algae, red algae, cyanobacteria, diatoms, yellow-green algae, golden algae, dinoflagellates, and seaweed. More specific examples of algae include algae of the genera Chaetoceros, Isochrysis, Pavlova, Pyramimonas, and Tisochrysis, with a preference for algae of the genus Chaetoceros, such as Chaetoceros gracilis. The algae in this disclosure may be a single species of algae or a population containing multiple species of algae. For example, a population of the same species of algae with genetic variation may be used. The algae of this disclosure may be algae derived from isolated algae. In the production methods of the embodiments, the algae may include algae of the genus Chaetoceros.

[0016] In this disclosure, the culture supernatant of algae is the liquid obtained by removing the solid components containing algal cells from a culture obtained by culturing algae in a liquid medium. Separation of the culture supernatant from the culture can be carried out by methods known to those skilled in the art, including filtration and / or centrifugation. The algal cells collected by centrifugation or the like can be used as feed for aquaculture.

[0017] In embodiments, the culture supernatant of algae may be the supernatant of a culture of algae grown in a closed culture facility, including a bioreactor, flask, culture tank, or other closed culture vessel. Alternatively, the culture supernatant of algae may be the culture supernatant of algae grown in an open culture facility, including an open pond. Depending on the properties of the control algae, the cultivation of algae may be carried out using a culture medium for algae known to those skilled in the art, seawater, other environmental water, diluted seawater, artificial seawater, mixtures thereof, or a solution having a partially common composition with these. The culture medium for algae can be any aqueous solution in which natural algae can grow without particular limitations, or an aqueous culture medium for algae with a specified composition may be used. The culture medium may be a culture medium for algae known to those skilled in the art, and may be a culture medium containing nutrients, a carbon source, rare metals, etc. Specific examples of the aqueous culture medium include IMK medium, SWM-3 medium, modified SWM-3 (mSWM-3) medium, modified media of these media, and media obtained by mixing these media or modified media. IMK medium contains 200mg / L NaNO3, 1.4mg / L Na2HPO4, 5mg / L K2HPO4, 2.68mg / L NH4Cl, 5.2mg / L Fe-EDTA, 0.332mg / L Mn-EDTA, 37.2mg / L Na2-EDTA, 0.023mg / L ZnSO4・7H2O, 0.014mg / L CoSO4・7H2O, 0.0073mg / L Na2MoO4・2H2O, 0.0025mg / L CuSO4・5H2O, 0.0017mg / L H2SeO3, 0.2mg / L Thiamin-HCl, 0.0015mg / L Biotin, 0.0015mg / L Vitamin B12 0.18mg / L A medium consisting of MnCl2・4H2O and the balance seawater. In particular, when culturing diatoms, 0.2–1 mM Na2SiO3 may be added to the IMK medium in addition to the above components.mSWM-3 medium contains 17 mg NaNO3, 1.56 mg NaH2PO4・2H2O, 5.68 mg Na2SiO3・9H2O, 1.12 mg Na2EDTA・2H2O, 0.084 mg Fe-EDTA, 0.0346 μg Na2SeO3, 1 ml P-1 metal solution (618.3 mg H3BO4, 69.25 mg MnCl2・4H2O, 5.45 mg ZnCl2, 238 μg CoCl2・6H2O, 100 ml distilled water), 0.2 μg Vitamin B12, 1 mL Vitamin mixture solution S3 (5 mg Thiamine HCl, 1 mg Nicotinic acid, 1 mg Calcium pantothenate, 0.1 mg p-Aminobenzoic acid, 0.01 mg Biotin, 50 mg Inositol, 0.02 mg Folic acid, 30 mg Thymine, 100 mL) The culture medium (pH 7.7-7.8) is prepared by mixing distilled water, 50 mg Tris(hydroxymethyl)aminomethane, and 98 mL of seawater. Algae cultivation may be carried out under natural light or under light irradiation. Algae cultivation may also be carried out by shaking.

[0018] In this disclosure, “isolation of low molecular weight compounds” may mean separating and purifying low molecular weight compounds contained in the culture supernatant of algae from the culture supernatant. Such isolation of low molecular weight compounds may involve pretreatment of the supernatant sample, including one or more of centrifugation, filtration, ultrafiltration, deproteinization, and solid-phase extraction (SPE).

[0019] Pre-treated or unpre-treated samples can be separated by high-performance liquid chromatography (HPLC). Columns suitable for HPLC include reversed-phase columns such as C18 columns, normal-phase columns such as silica columns, and gel filtration columns. Those skilled in the art will understand that these columns can be appropriately selected depending on the target small molecule. The mobile phase used in HPLC can also be appropriately selected by those skilled in the art depending on the column and the target small molecule. For example, kynurenine can be isolated and purified by separation using a reversed-phase column with water-acetonitrile (Sadok et al., Journal of Separation Science, 2017; Vol. 40, No. 15, pp. 3020-3045). Similarly, topiramate can be isolated and purified by separation using a reversed-phase column with water-acetonitrile or water-methanol as the mobile phase (Pint et al., Journal of Chromatographic Science, 2016, Vol. 54, No. 2, pp. 280-290). Detection in HPLC can be performed using detection methods known to those skilled in the art, such as mass spectrometry (MS), tandem mass spectrometry (MS / MS), ultraviolet-visible spectroscopy (UV-Vis), fluorescence spectroscopy (FL), and differential refractive index spectroscopy (ELS). Mass spectrometers used in MS and MS / MS include Fourier transform mass spectrometers (FT-MS) and time-of-flight mass spectrometers (TOF-MS).

[0020] In the manufacturing method of the embodiment, the culture supernatant of the algae may include, or may be, the culture supernatant obtained by culturing the algae under conditions including temperatures of 28°C to 35°C, 28°C to 32°C, 29°C to 31°C, or 30°C. When the algae are cultivated under conditions including temperatures of 28°C to 35°C, 28°C to 32°C, 29°C to 31°C, or 30°C, the low molecular weight compound may be one or more of the following: argininosuccinic acid, creatinine, ethanolamine, inosine, kynurenine, m-hydroxybenzoic acid, p-hydroxybenzoic acid, melamine, N-acetylputrescine, N 8 -acetylspermidine, piperidine, pyrophosphate, topiramate, β-hydroxyisovaleric acid, 2-hydroxyvaleric acid, 2-hydroxyisovaleric acid. These compounds are low molecular weight compounds that were detected only in the culture supernatant at 30°C in the examples.

[0021] Alternatively, when algae are cultured under conditions including temperatures of 28°C–35°C, 28°C–32°C, 29°C–31°C, or 30°C, the low molecular weight compounds may be any or more of the following: 1-methyladenine, 2'-deoxyadenosine, 5'-deoxyadenosine, 2'-deoxycytidine, 2'-deoxyguanosine, 2-hydroxyoctanoic acid, 2-oxoglutaric acid, 3-methylcytidine, 2'-O-methylcytidine, 3-sulfopropanediol, 4-guanidinobutyric acid, 4-hydroxyquinoline, 4-oxopyrrolidine-2-carboxylic acid, 5'-deoxy-5'-methylthioadenosine, 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylic acid, 5-methylcytidine, 7-methylguanine, adenine, adenosine, Ala, Ala-Ala, anthranilic acid, argininosuccinic acid, Asn, Asp, capryloylglycine, carnitine, choline, cis-aconitic acid, creatinine, cystathionine, cystine, cytidine, ethanolamine, galacturonic acid, glucuronic acid, Gln, Glu, gluconolactone, glutathione, GSSG, Gly, Gly-Asp, Gly-Leu, glycolic acid, guanine, guanosine, H-Asp(Gly-OH)-OH, homoserine, Ile, inosine, kynurenine, Leu, m-hydroxybenzoic acid, p-hydroxybenzoic acid, melamine, Met, methionine sulfoxide, mevalolactone, mucic acid,N-acetylgalactosamine, N-acetylglucosamine, N-acetylmannosamine, N-acetyllysine, N-acetylornithine, N-acetylputrescine, N, 1 ,N 12 -diethylspermine, N 1 -methylguanosine, N 2 -methylguanine, N 6 -methyladenine, N 6 -methyladenosine, N 8 -acetylspermidine, nicotinic acid, norspermidine, O-acetylcarnitine, ornithine, p-aminobenzoic acid, Phe, piperidine, Pro, pyridoxal, pyridoxine, pyrophosphate, Ser, Ser-Glu, spermidine, Thr, threonic acid, thymidine, thymine, topiramate, Trp, Tyr-Arg_divalent, uracil, uridine, urocanic acid, Val, β-hydroxyisovaleric acid, 2-hydroxyvaleric acid, 2-hydroxyisovaleric acid。

[0022] In the manufacturing method of the embodiment, the culture supernatant of the algae may include, or may be, the culture supernatant obtained by culturing the algae under conditions including temperatures of 20°C to 27°C, 23°C to 27°C, 24°C to 26°C, or 25°C. When the algae are cultivated under conditions including temperatures of 20°C to 27°C, 23°C to 27°C, 24°C to 26°C, or 25°C, the low molecular weight compounds may be one or more of the following: 1-methyladenosine, ATP, capryloylglycine, creatine, morpholine, Tyr-glu. These compounds are low molecular weight compounds that were detected only in the culture supernatant at 25°C in the example.

[0023] Alternatively, when algae are cultured under conditions including temperatures of 20°C–27°C, 23°C–27°C, 24°C–26°C, or 25°C, the low molecular weight compounds may be one or more of the following: 1-methyladenine, 1-methyladenosine, 2'-deoxyadenosine, 5'-deoxyadenosine, 2'-deoxycytidine, 2'-deoxyguanosine, 2-hydroxyoctanoic acid, 2-oxoglutaric acid, 3-methylcytidine, 2'-O-methylcytidine, 3-sulfopropanediol, 4-guanidinobutyric acid, 4-hydroxyquinoline, 4-oxopyrrolidine-2-carboxylic acid, 5'-deoxy-5'-methylthioadenosine, 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylic acid, 5-methylcytidine, 7-methylguanine, adenine, adenosine, Ala, Ala-Ala, anthranilic acid, Asn, Asp, ATP, capryloylglycine, carnitine, choline, cis-aconitic acid, creatine, cystathionine, cystine, cytidine, galacturonic acid, glucuronic acid, Gln, Glu, gluconolactone, glutathione, GSSG, Gly, Gly-Asp, Gly-Leu, glycolic acid, guanine, guanosine, H-Asp(Gly-OH)-OH, homoserine, Ile, Leu, Met, methionine sulfoxide, mevalolactone, morpholine, mucic acid, N-acetylgalactosamine, N-acetylglucosamine, N-acetylmannosamine, N-acetyllysine,N-acetylornithine, N-acetylputrescine, N, 1 ,N 12 -diethylspermine, N 1 -methylguanosine, N 2 -methylguanine, N 6 -methyladenine, N 6 -methyladenosine, nicotinic acid, norspermidine, O-acetylcarnitine, ornithine, p-aminobenzoic acid, Phe, Pro, pyridoxal, pyridoxine, Ser, Ser-Glu, spermidine, Thr, threonic acid, thymidine, thymine, Trp, Tyr-Arg, Tyr-Glu, uracil, uridine, urocanic acid, Val。

[0024] In the method for producing a low-molecular compound according to the embodiment, culturing may include culturing the algae for 8 days or more, 8 to 10 days, or 9 to 10 days. Alternatively, in the method for producing a low-molecular compound according to the embodiment, culturing may include culturing the algae for 1 to 4 days, 1 to 2 days, or 3 to 4 days.

[0025] In the manufacturing method of the embodiment, the culture supernatant of the algae may include, or may be, the culture supernatant obtained by culturing the algae for 8 days or more under conditions including a temperature of 20°C to 27°C, 23°C to 27°C, 24°C to 26°C, or 25°C. When the algae are cultivated for 8 days or more under conditions including a temperature of 20°C to 27°C, 23°C to 27°C, 24°C to 26°C, or 25°C, the low molecular weight compounds may be one or more of the following: 1-methyladenosine, 2'-deoxyguanosine, ATP, cystathionine, cytidine, gluconolactone, guanosine, N-acetylornithine, O-acetylcarnitine, ornithine, Pro, pyridoxine, Trp, Tyr-Glu. These compounds are low-molecular-weight compounds whose content in the culture supernatant of cultures cultured at 25°C for 8 days or more was greater than their content in the culture supernatant of cultures cultured at 30°C for 8 days or more in the examples.

[0026] Alternatively, when algae are cultured for 8 days or more under conditions including temperatures of 20°C–27°C, 23°C–27°C, 24°C–26°C, or 25°C, the low molecular weight compounds may be one or more of the following: 1-methyladenosine, ATP, ornithine, Tyr-Glu. These compounds were not detected in the culture supernatant of cultures cultured at 30°C for 8 days or more in the examples, but were detected only in the culture supernatant of cultures cultured at 25°C for 8 days or more.

[0027] In the production method of the embodiment, the culture supernatant of algae may contain, or may be, the culture supernatant obtained by culturing algae under conditions including a temperature of 28°C to 35°C, 28°C to 32°C, 29°C to 31°C, or 30°C for 8 days or more. When algae are cultured for 8 days or more under conditions including a temperature of 28°C to 35°C, 28°C to 32°C, 29°C to 31°C, or 30°C, the low-molecular-weight compound can be any one or more of the following: 2-hydroxyoctanoic acid, Ala, argininosuccinic acid, cis-aconitic acid, Glu, Gly-Leu, H-Asp(Gly-OH)-OH, homoserine, Ile, inosine, kynurenine, Leu, m-hydroxybenzoic acid, p-hydroxybenzoic acid, methionine sulfoxide, N-acetylgalactosamine, N-acetylglucosamine, N-acetylmannosamine, N-acetylputrescine, N 1 ,N 12 -diethylspermine, N 8 -acetylspermidine, norspermidine, Phe, pyrophosphate, spermidine, threonic acid, thymidine, topiramate, Val, β-hydroxyisovaleric acid, 2-hydroxyvaleric acid, 2-hydroxyisovaleric acid. These compounds are low-molecular-weight compounds whose contents in the culture supernatant of the culture cultured at 30°C for 8 days or more in the examples were greater than their contents in the culture supernatant of the culture cultured at 25°C for 8 days or more.

[0028] Alternatively, when algae are cultured for 8 days or more under conditions including temperatures of 28°C–35°C, 28°C–32°C, 29°C–31°C, or 30°C, the low molecular weight compounds may be one or more of the following: argininosuccinic acid, inosine, kynurenine, m-hydroxybenzoic acid, p-hydroxybenzoic acid, N-acetylputrescine, N 8 -Acetylspermidine, pyrophosphate, topiramate. These compounds were not detected in the culture supernatant of cultures cultured at 25°C for 8 days or more in the examples, but were detected only in the culture supernatant of cultures cultured at 30°C for 8 days or more. These are low-molecular-weight compounds.

[0029] The culture supernatant of algae may include, or may be, the culture supernatant obtained by culturing algae for 1 to 4 days under conditions including temperatures of 20°C to 27°C, 23°C to 27°C, 24°C to 26°C, or 25°C. When algae are cultivated for 1 to 4 days under conditions including temperatures of 20°C to 27°C, 23°C to 27°C, 24°C to 26°C, or 25°C, the low molecular weight compounds may be any or more of the following: 4-guanidinobutyric acid, capryloylglycine, creatine, glycolic acid, morpholine, urocanic acid.

[0030] The culture supernatant of algae may include, or may be, the culture supernatant obtained by culturing algae for 1 to 4 days under conditions including temperatures of 28°C to 35°C, 28°C to 32°C, 29°C to 31°C, or 30°C. When algae are cultivated for 1 to 4 days under conditions including temperatures of 28°C to 35°C, 28°C to 32°C, 29°C to 31°C, or 30°C, the low molecular weight compounds may be any or more of the following: 4-guanidinobutyric acid, ethanolamine, glycolic acid, urocanic acid.

[0031] The following examples illustrate the concept, but this disclosure is not limited to the examples described below.

[0032] IMK culture medium (Shioya MS Co., Ltd.) was dissolved in Daigo artificial seawater (Shioya MS Co., Ltd.), and approximately 1 x 10⁶ Chaetoceros gracilis, known as an algae feed for aquaculture, was added. 5 The cells / ml were added in the flask. Daigo artificial seawater does not contain organic components. Six of the above flasks were prepared and stored at 25°C and 30°C for 50 μmol photon / m³. 2 Three vials were cultured under light irradiation at 1 / s and shaking at 75 rpm.

[0033] Cell density increased under both 25°C and 30°C conditions up to day 9 of culture (Figure 1). This suggests that there was little difference in cell proliferation between 25°C and 30°C. 20 mL of culture medium was collected on days 2, 4, and 10 of culture, and cells were removed by centrifugation (16,000 g × 10 min). The supernatant was collected and filtered through a 0.2 μm pore filter, and the filtrate was collected.

[0034] The filtrate and IMK medium were subjected to metabolome analysis. 80 μL of supernatant was mixed with 20 μL of aqueous solution containing an internal standard (100 μM) and transferred to an ultrafiltration tube. This was then centrifuged (9,100 × g, 4°C, 60 min), followed by ultrafiltration, and then subjected to measurement. Metabolome measurements were performed using capillary electrophoresis-Fourier transform mass spectrometry (CE-FTMS) based on the conditions described in Japanese Patent No. 6106864. The relative amounts of each detected substance were calculated by dividing the peak area value by the area value of the internal standard and the sample volume.

[0035] Analysis revealed that 83 and 90 low-molecular-weight compounds, respectively, that were not detected in IMK medium alone (the culture medium for algae), were detected in the 25°C and 30°C samples on either day 2, 4, or 10 (Table 1).

[0036] Of these, 77 and 82 low-molecular-weight compounds were detected in the 25°C and 30°C samples on day 10, which is generally considered the harvest period for algae used as aquaculture feed (Table 2). Of these, 73 low-molecular-weight compounds were detected in both culture temperatures, but 10 of these compounds were detected in significantly higher relative amounts at 25°C, and 19 compounds were detected in significantly higher relative amounts at 30°C (Table 2). On the other hand, 4 and 9 compounds were detected only in the 25°C and 30°C samples, respectively (Table 2).

[0037] Furthermore, of the 77 and 82 compounds detected on day 10 at 25°C and 30°C, respectively, 35 and 41 compounds were not detected in the day 2 and day 4 samples at each culture temperature (Table 1). On the other hand, 6 and 4 compounds that were not detected on day 10 (25°C and 30°C samples, respectively) were detected in the day 2 or day 4 samples at each culture temperature (Table 3).

[0038] These substances contained numerous substances beneficial for aquaculture and health. Specifically, kynurenine, known as a stress-reducing substance in fish, and topiramate, known as a raw material for antiepileptic drugs, were detected in the culture supernatant after culturing at 30°C for 10 days. In addition, as shown in Table 4, several other beneficial substances were also detected.

[0039] The results above reveal that specific useful substances accumulate in the culture medium as Chaetoceros gracilis grows, and that its composition differs depending on the growth stage and culture temperature. In particular, it was suggested that the amount and type of accumulated substances can be controlled by adjusting the culture temperature. Furthermore, since useful substances were detected in the supernatant before the harvest season for use as aquaculture feed, it is possible to obtain useful substances that are not dependent on the harvest season by partially collecting the supernatant during the culture period.

[0040] According to the manufacturing method described herein, conventionally discarded algal feed production residue (culture supernatant) can be used to produce useful substances. Furthermore, by adjusting the timing of algal cell harvesting or supernatant collection, it becomes possible to manipulate the amount of useful substances obtained. For algae with a wide growth temperature range, the useful substances produced can be controlled by changing the culture temperature. At temperatures around room temperature, such as 25°C or 30°C, although the substances in the supernatant change, the growth rate of cells used as feed remains almost unchanged (Figure 1), which is advantageous for cell production. Furthermore, for algae grown in environments where the culture temperature fluctuates, such as outdoors, it becomes possible to predict the amount of useful substances in the supernatant by referring to the temperature during the culture and harvesting period.

[0041]

[0042]

[0043]

[0044]

[0045] This disclosure includes the following embodiments. [Item 1] A method for producing a low molecular weight compound, comprising isolating the low molecular weight compound contained in the culture supernatant of algae, wherein the low molecular weight compound is one or more of the following: kynurenine, topiramate, inosine, ornithine, ATP, carnitine, choline, cystine, Met, Thr, pyridoxal, pyridoxine, Trp, norspermidine, spermidine, Val, Leu, Phe, creatine, glycolic acid, capryloylglycine, 1-methyladenine, 1-methyladenosine, 2'-deoxyadenosine, 5'-deoxyadenosine, 2'-deoxycytidine, 2'-deoxyguanosine, 2-hydroxyoctanoic acid, 2-oxoglutaric acid, 3-methylcytidine, 2'-O-methylcytidine, 3-sulfopropanediol, 4-guanidinobutyric acid, 4-hydroxyquinoline, 4-oxopyrrolidine-2-carboxylic acid, 5'-deoxy-5'-methylthioadenosine, 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylic acid, 5-methylcytidine, 7-methylguanine, adenine, adenosine, Ala, Ala-Ala, anthranilic acid, argininosuccinic acid, Asn, Asp, cis-aconitic acid, creatinine, cystathionine, cytidine, ethanolamine, galacturonic acid, glucuronic acid, Gln, Glu, gluconolactone, glutathione, GSSG, Gly, Gly-Asp, Gly-Leu, guanine, guanosine,H-Asp(Gly-OH)-OH, homoserine, Ile, m-hydroxybenzoic acid, p-hydroxybenzoic acid, melamine, methionine sulfoxide, mevalolactone, morpholine, mucic acid, N-acetylgalactosamine, N-acetylglucosamine, N-acetylmannosamine, N-acetyllysine, N-acetylornithine, N-acetylputrescine, N, 1 ,N 12 -diethylspermine, N 1 -methylguanosine, N 2 -methylguanine, N 6 -methyladenine, N 6 -methyladenosine, N 8-acetylspermidine, nicotinic acid, O-acetylcarnitine, p-aminobenzoic acid, piperidine, Pro, pyrophosphate, Ser, Ser-Glu, threonic acid, thymidine, thymine, Tyr-Arg, Tyr-Glu, uracil, uridine, urocanic acid, β-hydroxyisovaleric acid, 2-hydroxyvaleric acid, 2-hydroxyisovaleric acid. [Claim 2] The method according to claim 1, wherein the algae comprises algae of the genus Chaetoceros. [Claim 3] The method according to claim 1 or 2, wherein the culture supernatant comprises a culture supernatant obtained by culturing the algae under conditions including a temperature of 28°C to 35°C. [Claim 4] The method according to claim 3, wherein the culturing comprises culturing the algae for 8 days or more. [Claim 5] The method according to claim 1 or 2, wherein the culture supernatant comprises a culture supernatant obtained by culturing the algae under conditions including a temperature of 20°C to 27°C. [Claim 6] The method according to claim 5, wherein the culturing comprises culturing the algae for eight days or more. [Claim 7] The method according to claim 1 or 2, wherein the culturing comprises culturing the algae for one to four days. [Claim 8] The method according to claim 7, wherein the culture supernatant comprises a culture supernatant obtained by culturing the algae under conditions including a temperature of 28°C to 35°C. [Claim 9] The method according to claim 1 or 2, wherein the culturing comprises culturing the algae for one to four days. [Claim 10] The method according to claim 9, wherein the culture supernatant comprises a culture supernatant obtained by culturing the algae under conditions including a temperature of 20°C to 27°C.[Item 11] A method for producing a low molecular weight compound, comprising isolating the low molecular weight compound contained in the culture supernatant of algae, wherein the culture supernatant includes a culture supernatant obtained by culturing the algae under conditions including a temperature of 28°C to 35°C, wherein the culturing comprises culturing the algae for 8 days or more, and the low molecular weight compound is one or more of the following: kynurenine, topiramate, inosine, argininosuccinic acid, m-hydroxybenzoic acid, p-hydroxybenzoic acid, N-acetylputrescine, N. 8 -Acetylspermidine, pyrophosphate. [Claim 12] The method according to claim 11, wherein the algae comprises algae of the genus Chaetoceros. [Claim 13] The method according to claim 11 or 12, wherein the algae comprises Chaetoceros gracilis.

[0046] While this disclosure has been described with reference to several embodiments described above, this disclosure is not limited to the examples given in these embodiments. Various modifications can be made to the configuration and details of the present invention within the scope of this disclosure.

Claims

1. A method for producing a low molecular weight compound, comprising isolating the low molecular weight compound contained in the culture supernatant of algae, wherein the low molecular weight compound is one or more of the following: kynurenine, topiramate, inosine, ornithine, ATP, carnitine, choline, cystine, Met, Thr, pyridoxal, pyridoxine, Trp, norspermidine, spermidine, Val, Leu, Phe, creatine, glycolic acid, capryloylglycine, 1-methyladenine, 1-methyladenosine, 2'-deoxyadenosine, 5'-deoxyadenosine, 2'-deoxycytidine, 2'-deoxyguanosine, 2-hydroxyoctanoic acid, 2-oxoglutaric acid, 3-methylcytidine, 2'-O-methylcytidine, 3-sulfopropanediol, 4-guanidinobutyric acid acid, 4-hydroxyquinoline, 4-oxopyrrolidine-2-carboxylic acid, 5'-deoxy-5'-methylthioadenosine, 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylic acid, 5-methylcytidine, 7-methylguanine, adenine, adenosine, Ala, Ala-Ala, anthranilic acid, argininosuccinic acid, Asn, Asp, cis-aconitic acid, creatinine, cystathionine, cytidine, ethanolamine, galacturonic acid, glucuronic acid, Gln, Glu, gluconolactone, glutathione, GSSG, Gly, Gly-Asp, Gly-Leu, guanine, guanosine, H-Asp(Gly-OH)-OH,homoserine, Ile, m-hydroxybenzoic acid, p-hydroxybenzoic acid, melamine, methionine sulfoxide, mevalolactone, morpholine, mucic acid, N-acetylgalactosamine, N-acetylglucosamine, N-acetylmannosamine, N-acetyllysine, N-acetylornithine, N-acetylputrescine, N, 1 ,N 12 -diethylspermine, N 1 -methylguanosine, N 2 -methylguanine, N 6 -methyladenine, N 6 -methyladenosine, N 8 -acetylspermidine, nicotinic acid, O-acetylcarnitine, p-aminobenzoic acid, piperidine, Pro, pyrophosphate, Ser, Ser-Glu, threonic acid, thymidine, thymine, Tyr-Arg, Tyr-Glu, uracil, uridine, urocanic acid, β-hydroxyisovaleric acid, 2-hydroxyvaleric acid, 2-hydroxyisovaleric acid。 2. The method according to claim 1, wherein the algae include algae of the genus Chaetoceros.

3. The method according to claim 1 or 2, wherein the culture supernatant includes a culture supernatant obtained by culturing the algae under conditions including a temperature of 28°C to 35°C.

4. The method according to claim 3, wherein the cultivation includes culturing the algae for eight days or more.

5. A method for producing a low molecular weight compound, comprising isolating the low molecular weight compound contained in the culture supernatant of algae, wherein the culture supernatant includes the culture supernatant obtained by culturing the algae under conditions including a temperature of 28°C to 35°C, wherein the culturing comprises culturing the algae for 8 days or more, and the low molecular weight compound is one or more of the following: kynurenine, topiramate, inosine, argininosuccinic acid, m-hydroxybenzoic acid, p-hydroxybenzoic acid, N-acetylputrescine, N 8 -Acetylspermidine, pyrophosphate.

6. The method according to claim 5, wherein the algae include algae of the genus Chaetoceros.

7. The method according to claim 5 or 6, wherein the algae includes Chaetoceros gracilis.

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